Acid Resistence in Escherichi coli and Shigella species A hallmark of bacillary dysentery is the fact that as few as 10 bacteria are sufficient to cause disease. In contrast the infective dose for Salmonella gastritis is greater than 100,000 bacteria whereas over a million Vibrio cholera are required to cause cholera. Aside from shigellosis, the only other bacterial diarrhea associated with such low infectious dose is that caused by strains of Escherichia coli such as the "MacDonald hamburger" strains (E. coli 0157). These strains are among a group of newly emerging infectious agents which have become increasingly important both as diarrheal agents and as a cause of a more serious disease, HUS, or hemolytic uremic syndrome. A major focus of our research is to determine how Shigella and enterohemorrhagic E. coli are able to pass through the stomach with a pH of less than 3.0 in order to reach the intestines. The hypothesis underlying this work is that the ability of Shigella species to survive exposure to the extreme acid environment of the stomach contributes to the low infective dose. Work from our laboratory had established the fact that acid resistance was induced during stationary phase, and required the activity of the stationary sigma factor, RpoS. Survival to extreme pH in ES does not require prior acid challenge though it is growth phase dependent. Since more acid sensitive bacteria such as Salmonella species express RpoS, we have focused on identifying RpoS-dependent genes unique to ES. Using transposon mutagenesis we have identified a number of genes--- gadC, gadB and hdeA/B which are required for acid resistance. Deletion derivations of rpoS and gadC were extremely acid sensitive confirming the role of these genes in acid resistance. Using Northern blots, it was possible to determine that the induction of gadC occurred during stationary phase and was rpoS dependent. However, DNA from gadC hybridized with two transcripts; one of these appeared to be the gadC transcript, whereas fainter hybridization was obtained with a larger transcript. An analysis of a gadC containing cosmid from the E. coli genome project, showed the presence of three contiguous open reading frames. The first of these encodes gadB, which encodes a glutamate decarboxylase, the second encodes gadC described above, and the third open reading frame was designed gadD. a consensus sequence typical of rpoS-regulated genes. Although glutamate decarboxylases are very common among eucaryotes, they are extremely rare in procaryotes. In order to ascertain the distribution of gadB and gadC among enteric bacteria, Southern hybridization was performed using specific gadB and gadC probes hybridized against DNA from all major representative Enterbacteriacae as well as against several Pseudomonas species. Results from these studies showed that gadC and gadB had a similar distribution and were found only is Shigella species and E. coli. Until very recently, the only genetic evidence for the gad operon in bacteria was data from EC. However, recently, a paper by Sanders et al. (1998, Mol. Microbiol. 27, 299-310) has shown that this operon is also present in the gram positive homofermentative organism, L. lactis. As in ES, the gad operon in L. lactis protects against exposure to low acid and is most highly expressed by resting cells. In addition, glutamate decarboxylase activity has been reported from a number of anaerobes which are part of normal human intestinal flora such as Bacteroides species, Clostridia species and Eubacteria limosum. The presence of this operon in a number of species which are metabolically limited by their inability to obtain energy via oxidative processes, suggests a second possible function for the gad operon. Work by Higuchi ((1997, J. Bacteriol. 175, 2864- 2870) on Lactobacilli has shown that decarboxylation of glutamate is accompanied by production of ATP. In light of this knowledge, we have proposed a second model for the function of gadBC. In this model (Figure 2) glutamate 2- is transported into the cell where it is converted to g-aminobutyrate1-. . Transport of g-aminobutyrate out of the cell via the antiporter GadC results creates an electrogenic gradient (DY) which can be used to generate ATP via the proton pump FoF1. Thus the gad operon might function both to protect bacteria in transit through the stomach where they are exposed to low pH, as well as to provide ES with an additional source of metabolic energy during anaerobic growth in the gut.